A way to understand the function of a gene within an organism is to inhibit its expression. Inhibition of gene expression can be accomplished, for example, by interrupting or deleting the DNA sequence of the gene, resulting in “knock-out” of the gene (Austin et al., Nat. Genetics 36:921-924). Gene knock-outs mostly have been carried out through homologous recombination (HR), a technique applicable across a wide array of organisms from bacteria to mammals. Another way for studying gene function can be through genetic “knock-in”, which is also usually performed by HR. HR for purposes of gene targeting (knock-out or knock-in) can employ the presence of an exogenously supplied DNA having homology with the target site (“donor DNA”).
HR for gene targeting has been shown to be enhanced when the targeted DNA site contains a double-strand break (Rudin et al., Genetics 122:519-534; Smih et al., Nucl. Acids Res. 23:5012-5019). Strategies for introducing double-strand breaks to facilitate HR-mediated DNA targeting have therefore been developed. For example, zinc finger nucleases have been engineered to cleave specific DNA sites leading to enhanced levels of HR at the site when a donor DNA was present (Bibikova et al., Science 300:764; Bibikova et al., Mol. Cell. Biol. 21:289-297). Similarly, artificial meganucleases (homing endonucleases) and transcription activator-like effector (TALE) nucleases have also been developed for use in HR-mediated DNA targeting (Epinat et al., Nucleic Acids Res. 31: 2952-2962; Miller et al., Nat. Biotech. 29:143-148).
Loci encoding CRISPR (clustered regularly interspaced short palindromic repeats) DNA cleavage systems have been found exclusively in about 40% of bacterial genomes and most archaeal genomes (Horvath and Barrangou, Science 327:167-170; Karginov and Hannon, Mol. Cell 37:7-19). In particular, the CRISPR-associated (Cas) RNA-guided endonuclease (RGEN), Cas9, of the type II CRIPSR system has been developed as a means for introducing site-specific DNA strand breaks that stimulate HR with donor DNA (WO2015/026883, published Feb. 26, 2015. The sequence of the RNA component of Cas9 can be designed such that Cas9 recognizes and cleaves DNA containing (i) sequence complementary to a portion of the RNA component and (ii) a protospacer adjacent motif (PAM) sequence.
Native Cas9/RNA complexes comprise two RNA sequences, a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). A crRNA contains, in the 5′-to-3′ direction, a unique sequence complementary to a target DNA site and a portion of a sequence encoded by a repeat region of the CRISPR locus from which the crRNA was derived. A tracrRNA contains, in the 5′-to-3′ direction, a sequence that anneals with the repeat region of crRNA and a stem loop-containing portion. Recent work has led to the development of guide RNAs (gRNA), which are chimeric sequences containing, in the 5′-to-3′ direction, a crRNA linked to a tracrRNA (WO2015/026883, published Feb. 26, 2015.
Protein and RNA components for performing Cas9-mediated DNA targeting in a cell have been provided in some studies through recombinant DNA expression strategies. For example, Cas9 protein has been expressed in cells using nucleic acid-based expression systems. Methods of expressing RNA components such as gRNA in certain cell types have included using RNA polymerase III (Pol III) promoters, which allow for transcription of RNA with precisely defined, unmodified, 5′- and 3′-ends (DiCarlo et al., Nucleic Acids Res. 41: 4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3:e161). These protein and RNA expression techniques have been applied in cells of several different species including maize and soybean (WO2015/026883, published Feb. 26, 2015, as well as humans, mouse, zebrafish, Trichoderma and Saccharomyces cerevisiae. 
Despite these advances, other means of providing protein and RNA components in a cell, such as a microbial cell, to mediate Cas9-mediated DNA targeting are of interest.